Coleopterans are a significant group of agricultural pests that cause extensive damage to crops each year. Examples of coleopteran pests include corn rootworm and alfalfa weevils. Additional notable examples include Colorado potato beetle, boll weevil, and Japanese beetle.
Insecticidal crystal proteins from some strains of Bacillus thuringiensis (B.t.) are well-known in the art. See, e.g., Höfte et al., Microbial Reviews, Vol. 53, No. 2, pp. 242-255 (1989). These proteins are typically produced by the bacteria as approximately 130 kDa protoxins that are then cleaved by proteases in the insect midgut, after ingestion by the insect, to yield a roughly 60 kDa core toxin. These proteins are known as crystal proteins because distinct crystalline inclusions can be observed with spores in some strains of B.t. These crystalline inclusions often comprise a mixture of distinct proteins.
An entirely new insecticidal protein system was discovered in Bacillus thuringiensis as disclosed in WO 97/40162. Unlike the 130 kDa-type protoxins, this new system comprises two proteins—one of approximately 15 kDa and the other of about 45 kDa. See also U.S. Pat. No. 6,083,499; U.S. Pat. No. 6,127,180; Moellenbeck et al., Nature Biotechnology 19:668-672 (2001); and Ellis et al., Applied and Environmental Microbiology 68:1137-1145 (2002). These proteins have now been assigned to their own classes, and accordingly received the Cry designations of Cry34 and Cry35, respectively. See Crickmore et al. website (biols.susx.ac.uk/home/Neil_Crickmore/Bt/). Many other proteins of this type of system have now been disclosed. See e.g. U.S. Pat. No. 6,372,480; WO 01/14417; and WO 00/66742. Plant-optimized genes that encode such proteins, wherein the genes are engineered to use codons for optimized expression in plants, have also been disclosed. See e.g. U.S. Pat. No. 6,218,188 and WO 00/24904.